Quantitative PCR has been used to measure the relative levels of RNA and DNA from a variety of different samples. It is possible, at times, to find a direct correlation between the amount of starting target material and the amount of PCR product (Mullis and Faloona, Methods Enzymol. 155:335-50 (1987); Ferre, F., PCR Methods Applic. 2:1-9 (1992); Sardelli, A. D., Amplifications: A Forum for PCR Users (9):1-5 (1993)). However, this is often not the case for clinical samples due to the presence of inhibitors of PCR in samples and differing efficiencies in sample recovery and kinetics of PCR (Holodniy et al., J. Clin. Micro. 29:676-679 (1991); Beutler et al., BioTechniques 9:166 (1990); Franchis et al., Nucl. Acids Res. 16:10355 (1988); Coutlee et al., J. Infect. Dis. 164:817-818 (1991)). The use of PCR as a quantitative assay often presents problems. For instance, small variations in amplification efficiency can change the yield of the product and make it difficult to accurately estimate the amount present in the starting material (Gilliland et al., Proc. Natl. Acad. Sci. USA 87:2725-2729 (1990)). To avoid these problems, several laboratories have described the use of internal standards in PCR (Gilliland et al., Proc. Natl. Acad. Sci. USA 87:2725-2729 (1990); Wang et al., Proc. Natl. Acad. Sci. USA 86:9717-9721 (1989); Becker-Andre et al., Nucl. Acids Res. 17:9437-9446 (1990); Chelly et al., Eur. J. Biochem. 187:691-698 (1990); Bergenhem et al., Proc. Natl. Acad. Sci. USA 89:8798-8802 (1992); Aoki-Sei et al., AIDS Res. Human Retro. 8:1263-1270 (1992); Siebert et al., Nature 359:557-558 (1992)). Generally, the internal standard DNA or RNA share the same primers as the target DNA or RNA, but will contain either a deletion or an insertion so that the products obtained from the standard and target can be distinguished.
A variation in the use of an internal control is the competitive PCR procedure (Gilliland et al., Proc. Natl. Acad. Sci. USA 87:2725-2729 (1990)). In this method, varying known amounts of the internal standards are added to equal aliquots of the sample containing the unknown target sequence. The internal standard and the target sequence compete equally for primer binding and amplification in the PCR. Variables such as the efficiency of amplification and the number of cycles will have the same effect on both templates. Equal amounts of products will be formed when the initial concentrations of the templates are equal. Experimentally, the ratio of products formed can be determined and the equivalence point can be calculated. This method has been successfully employed to quantify the amount of HIV-1 RNAs in clinical samples (Bagnarelli et al., J. Med. Virol. 34:89-95 (1991); Menzo et al., J. Clin. Microbiol. 30:1752-1757 (1992)). However, this method does not control the variations in RNA recovery from sample to sample. Similarly, viral DNA isolation is often incomplete and control of the amount of DNA would be of great use.
In the present invention, the inventors have developed a modified method in which an infectious tagged virus is used as the source of competitor nucleic acid. The tagged virus is a mutant or variant of the virus suspected of being in the sample. Different amounts of the tagged virus can be added to equal aliquots of the sample containing an unknown amount of virus, followed by nucleic acid extraction and amplification carried out in a manner that allows for relatively precise quantitation of the amount of viral nucleic acid present in the sample.